{8 N-Me-Ile{40 ,Ile{hu 8{b {9 -Angiotensin II as an angiotensin antagonist

ABSTRACT

A new octapeptide having profound angiotensin antagonist properties has been found. The new compound resembles angiotensin II except for the two terminal amino acids, carrying an unnatural amino acid at the N-terminus.

United States Patent [191 Bumpus et al.

1 1 Dec. 2, 1975 1 [N-MEJLE',ILE I-ANGIOTENSIN 11 AS AN ANGIOTENSINANTAGONIST [22] Filed: May 10, 1974 [21] Appl. No.: 468,831

[52] US. Cl 260/112Q5; 424/177 [51] Int. Cl. C07C 103/52; A61K 37/00[58] Field of Search 260/112.5

[56] References Cited UNITED STATES PATENTS 3,816,385 6/1974 Gillessenet al 260/112.5

Primary ExaminerLewis Gotts Assistant Examiner-Regina1d J. SuyatAttorney, Agent, or Firm-Paul D. Burgauer; Robert L. Niblack [57]ABSTRACT A new octapeptide having profound angiotensin antagonistproperties has been found. The new compound resembles angiotensin 11except for the two terminal amino acids. carrying an unnatural aminoacid at the N-terminus.

1 Claim, N0 Drawings [N-ME-ILE'JLE ]-ANGIOTENSIN II AS AN ANGIOTENSINANTAGONIST DETAILED DESCRIPTION OF THE INVENTION This invention relatesto polypeptides. More particularly, it is concerned with the octapeptideof the formula:

L-(N Methyl)-Ile-L-Arg-L-Val-L-Tyr-L-Ile-L-His-L-Prc-L-lle The peptideof this invention possesses valuable pharmacological activity. It iscapable of inhibiting the pressor effect of angiotensin II. Thus, whenadministered by intravenous infusion into rats in a very small amount,the pressor effect of angiotensin II is inhibited. By virtue of thisinhibitory property upon angiotensin II induced blood pressureelevation, the peptide of this invention is a valuable agent forcounteracting hypertension due to angiotensin II. It is also capable ofreducing blood pressure in acute unilateral renal hypertensive rats uponintravenous infusion.

The octapeptide of this invention is readily prepared in accordance withknown methods for preparing peptides. Such methods involve the buildingof a linear chain of amino acids through repetitive amide linkagesemploying in such sequential alignment the.necessary protective groupssusceptible of ready removal by conventional cleavage methods which donot affect the peptide bonds. The adaptation of such methods to thepeptide of this invention is described in the example below. All theamino acids used in the above structure of Formula I are in theL-configuration. The corresponding chain with D- amino acids does notshow the pharmacological effect described below.

In order to illustrate the method for making the compound of structureI, reference is made to the following example, which however, is notintended to limit the invention in any respect. In this example, theabbreviation BOC- is .used in its accepted meaning, referring totertiary butyloxycarbonyl. It also should be understood that all aminoacids used in this example are in their L- configuration. The BOC- aminoacids used below are either commercially available or were preparedaccording to the method of Schwyzer et al., Helv. Chim. Acta., 42, 2622(1959).

EXAMPLE A solution of 4.6 g. of BOC-isoleucine and 2.8 ml. oftriethylamine in 25 ml. of ethanol was added to 20 g. ofchloromethylpolystyrene/divinylbenzene (98:2) copolymer of a mesh sizebetween 200 and 400, containing 5.02% chlorine. The mixture was stirredat C. for 36 hours. The esterified polymer was filtered, washed severaltimes, in sequence, with ethanol, dilute acetic acid, water, ethanol andmethanol. The polymer was dried in vacuo over phosphorus pentoxide.Hydrolysis of an aliquot of the polymer and subsequent amino acidanalysis indicated that 0.4 millimoles of BOC- isoleucine wereesterified per gram of the polymer. Further coupling of the BOC-proline,BOC-N- imidazole-benzyl-histidine, BOC-isoleucine, BOC-(O-benzyl)-tyrosine, BOC-valine and BOC-nitroarginine in the respectiveorder was carried out by utilizing the action given below for each aminoacid residue. Unless specified, all washings were carried out threetimes for three minutes each, first with glacial acetic acid and secondwith methylene chloride. The BOC group was removed by treatment with 40%(volume/volume) of trifluoroacetic acid in methylene chloride for 30minutes, preceded by a prewash with this reagent for 3 minutes to avoiddilution of the trifluoroacetic acid by the previous methylene chloridewash. The deprotected amino acid polymer ester was washed five times for3 minutes each with chloroform and the trifluoroacetate salt neutralizedby treating the residue for 7 minutes with 10% triethylamine inchloroform, followed by three 3-minute washings with chloroform andmethylene chloride in sequence. The subsequent incoming BOC-amino acidwas added in a 2-fold excess in methylene chloride and the mixture wasstirred for 10 minutes. In the case of BOC-nitroarginine and BOC- benzylhistidine, these materials were first dissolved in dimethylformamide,followed by filtration and mixing the filtrate with one-third volume ofmethylene chloride. Both of these derivatives were used in 3-foldexcesses. Coupling was aided in each instance by the addition of a2-fold excess of dicyclohexylcarbodiimide in methylene chloride andmixing was carried'out for 2.5 hours except in the coupling stepsinvolving Arg or His where DCl was used in a 3-fold excess and mixingallowed for eight hours. The polymer-peptide chain was then washed withDMF followed by a washing with DMF/methylene chloride (1:1) and thecoupling step with the BOC-amino acid and DC] was repeated using amixture of 1:1 DMF/methylene chloride as the solvent. The polymer chainwas then washed with methanol to remove diiiyclohexylurea and finallywashed with DMF. Completeness of each coupling at intermediate stageswas checked by known color reaction tests. The apparatus used for theabove synthesis was of the manual type described by Khosla, Smeby andBumpus, Science, 156, 253 (1967). All couplings were carried out at 0 5C. to avoid racemization; l-hydroxybenzotriazole was used as an additiveto minimize racemization of histidine during the coupling ofBOC-imidazolebenzyl histidine [see G. C. Windridge and E. C. Jorgensen,J.A.C.S., 93, 6318 (1971)].

In the above sequence, the blocked (protected) amino acids were coupledin sequence to the isoleucine-polymer, using BOC-proline,BOC-N-imidazolebenzyl-histidine, BOC-isoleucine, BOC-(O-benzyl)- 3tyrosine, BOC-valine and BOC-nitroarginine to produce a peptide of thestructure of formula I with amino acids 3 8 bound to the polymersubstrate.

BOC-N-methylisoleucine was prepared by a modified procedure of Olsen (J.Org. Chem. 35, 1912 [1970]), as previously reported for BOC-N-methylphenylalanine (Khosla et al., J.A.C.S. 94, 4721 [1972]), except that CHl was added in 3 lots over a period of 50 hours. BOC-N-methylisoleucinewas then coupled to 4 g. of the above heptapeptide resin ester by thegeneral procedure described above using dicyclohexylcarbodiimide as thecoupling reagent and methylene chloride as the coupling medium. Afterstirring for 3 hours, the mixture was filtered, washed with DMF and thiscoupling step was repeated.

The protected peptide polymer was suspended in approximately 100 ml. offreshly distilled trifluoroacetic acid and a slow stream of hydrogenbromide, prewashed with 10% resorcinol in acetic acid, was passedthrough the suspension under anhydrous conditions for about 30 minuteswith occasional shaking. The suspension was filtered and the polymer waswashed with trifluoroacetic acid. The combined filtrates were evaporatedat room temperature in vacuo. The amorphous powder was washed withether, dissolved in a mixture of methanol/acetic acid/water 101111 andthe solution hydrogenated at 3.5 kg./cm. over 0.5 parts of palladiumblack per part of peptide weight for 48 hours with shaking. The productwas purified on a 5 X 80 cm. column of Sephadex G 25 (a partiallycross-linked dextran gel having an exclusion of molecular weight sizesof 5000, marketed by Pharmacia of Uppsala, Sweden) usingn-butanol/pyridine/water (:2:5) as the developing solvent. The averageyield of the product obtained in this manner varied between 40 and 60percent based on the millimoles of C-terminal amino acid esterified ontothe polymer. Fractions in column chromatography were cut without regardfor yield to obtain the desired compound in the pureform and no attemptwas made to rechromatograph the minor fractions for identificationpurposes. The homogeneity of the compound was determined by thin-layerchromatography in various solvent systems of different pH,electrophoresis at pH 1.95 and 8.6 and amino acid analysis, proving thatthe compound is homogeneous with R, 0.75 (nbutanol/acetic acid/water421:5) and R, 0.86 (nbutanol/ethylacetatelacetic acid/water 1:l:l:1), R,0.48 (n-butanol/pyridine/water 10125), R; 0.89 (nbutanol/aceticacid/water/pyridine :3:12110) on cellulose thin-layer plates. Thechemical analysis showed that the required amino acids were present inthe expected ratio.

The above antagonist was studied both in vitro and in vivo. The in vitroassay mainly evolves r pund an assay of isolated rabbit aortic stripsmounted Ta muscle bath in 5 ml. of Krebs solution. 3.

The strips were placed under 2 grams'liof passive tension and allowed toequilibrate for 2 hours. Cumulative dose-response curves were obtainedfor angiotensin II at concentrations of l to 64 ng./ml. The antagonistwas added to the bath at a certain concentration, allowed to equilibratefor 5 minutes and the response to angiotensin II again tested. Thevarious doses of the antagonist thus tested were 1, 10, 100 and 1,000

ng./ml. Changes in these curves were plotted and measurement of shift tothe right of the doseresponse curve were measured. At the same time, itwas assured that the maximal response of the tissue remained unchanged.At the end of the experimental period, the an tagonist was washed outand cumulative dose-response curves were repeated again. Results ofthese studies are expressed in the form of pA values as defined by O.Arunlakshna and H. O. Schild, Brit. J. Pharmacol, 14, 48 (1959). Thecompound of formula I showed a pA value of 8.73 i 0.09.

In the in vivo assays, rats were anesthetized with sodium amytal andfurther treated with 0.6 mg. atropine and pentolinium tartrate. Theywere vagotomized and direct recording of the blood pressure was measuredthrough the carotid artery, while the femoral artery was used forinfusing the desired dose over a period of 30 minutes in a physiologicalsaline solution.

The dose-response curves of angiotensin II at 0.9 to 54 ng./kg./min.were compared by linear regression analysis with those of the antagonistat doses of 1, 10, and 1000 ng./kg./min. Infusion of the aboveantagonist into rats thus prepared led to the initial transient pressorresponse of 0.74 on a scale set artificially at 100 for angiotensin IIusing the same system.

For determining its antagonistic properties the compound was infusedinto rats at a dose-level of 250, 500, 1250 ng./kg./min. The blockingeffect of the compound was calculated from the dose-response curve ofangiotensin II before and during the infusion of the analog. Generalprocedure adopted for this assay has been described by Bumpus et al.,Circ. Res. 32-33 (Supplement I), I-1 50 ('1973). The compound blockedthe pressor response of angiotensin II at a dose-level of 250ng./kg./min. or greater.

Since the above compound is particularly suitable for injection orinfusion, it is particularly valuable that the compound iswater-soluble. A suitable dosage unit can be prepared by simplydissolving the above compound in water or physiological saline at aconcentration of between 50 and 6,000 ng./ml. Such a solution can beadministered directly or it can be stored under proper conditions forperiods of several weeks without deterioration, particularly whencombined with l 5 percent of a preservative such as benzyl alcoholand/or is buffered to a suitable pH with a nontoxic, pharmaceuticallyacceptable buffer. A commonly employed buffer for an injectable solutionis tris(hydroxymethyl)aminomethane but simple salts such as sodiumphosphate or acetate can be used. Preferably, the vehicle or medium inwhich the compound of formula I is dissolved for an injectable orinfusable solution is buffered to a pH of 7 to 7.5.

The duration of action of the compound can be prolonged by injecting anoil solution intramuscularly. Oils suitable for this purpose are codliver oil, sesame oil, refined coconut oil, etc. Antagonistic effectsare thus observed for 24 hours or longer after a single i.m. injectionof the drug dissolved in such an oil.

What is claimed is:

l. The octapeptide L-(N-methyl)Ile-L-Arg-L-Val-L-Tyr-L-Ile-L-His-L-Pro-L-Ile.

1. THE OCTAPEPTIDEL-(N-METHYL)LLE-L-ARG-L-VAL-L-TYR-LLLE-L-HIS-PRO-L-ILE.